Indian Journal of Experimental Biology Vol. 42, September 2004, pp. 884-892

Oxyradicals under UV -B stress and their quenching by antioxidants

Karishma Jain, Sunita Kalaria & K N Guruprasad*

School of Life Sciences, Oevi Ahilya University , Khandwa Road, Indore 452 017, India

Received 24 October 2003; revised 12 April 2004

Formation of oxyradicals under UV-B stress was investigated using cucumber cotyledons. UV-il radiation induced production of free radical s which were analyzed by ESR spectroscopy. Evidence was obtained for the formation of superoxide and hydroxyl radicals in the ti ssues by comparing PBN-adducts formed with radicals obtained by chemical auto­oxidation of KO] and Fenton's reaction. Addition of superoxide dismutase (SOD) to the rcacti( .n mixture partially reduced the intensity of signals confirming the production of superoxide radical as well as hydroxyl radical s. These radicals were quenched ill vitro by the natural antioxidants a-tocopherol , ascorbic ac id and benzoquinone. Changes in the level of antioxidants were also monitored under UV-il stres~. The endogenous level of ascorbic acid was enhanced and a-tocopherol level was reduced in the tissue after exposure to UV -il radiation. The present report happens to be the first direct evidence obtained for the formation of superoxide and hydroxyl radicals in platH tissues exposed to UV-il radiation.

Keywords: Ascorbic acid, Benzoquinone, Cucllmber cotyledons, Superoxide radical , I-Jydro-oxyradical , a-Toco~'herol ,

UV-il stress.

Studies on biological effects of UV-B radiation have remained in fOCll'> in recent years since the depletion of ozone by pollutants like CFCs and oxides of nitrogen are known to cause spectral radiation changes in the region of UV-B (280-320 nl11)I ,2, Several studies have been undertaken to observe the physiological changes in plants at enhanced levels of UV -B radiation by supplementing natural solar radiation or artificial light sources with UV-13 radiation. These studies have indicated the deleterious effects of UV -B radiation on growth of the plants and the rate of photosynthesis resulting in reduced biomass in several crop plants like wheat, rice, sorghum, maize and soybean3

-6

. Most of the deleterious effects of UV -B radiation are related to the formation of free radicals and resulting oxidative stress 7-10. Molecular studies have revealed that UV-B exposure also results in DNA damage, decline in RNA, enzyme activity and induction of synthesis of pathogenesis related proteins 11-13.

Active oxygen species [AOS] are routinely generated in low levels in non-stressed plant cells in chloroplast, mitochondria and also by membrane bound exocellular enzymes involved in redox reactions. The concentration of AOS is known to increase under chemical and environmental stress

*Correspondent author: Email : knguruprasad@hotmail.com; Phone: 91-731-2467029; Fax: 91-731-2360026

conditions. Increased production of AOS has been reported under diverse environmental stresses like I 'll' 14 I I 15 d ' . 16 f1 d' 17 c 11 lIlg; ( roug 1t -; eSlccatlon; 00 lt1g ;

f . 18 . b' I . f . 1'1 d' l) I reezlllg ; micro 1U lllectlOn; WOl"1 mg anc exposure to UV_B 20. Environmental chemical stress caused by the release of metal iOIlS, sulphur dioxide (S02), nitrogen oxides (NOx) and ozone (03) are also associated with the formation of reactive oxygen species21 . Reactive oxygen species include singlet oxygen, superoxide radical, hydrogen peroxide and hydroxyl radical.

Free radicals generated by various oxidative stress cause changes in the unsaturated fatty acids that affect the structural and functional properties of cell membranes. Changes in the malondialdehyde content, an indicator of lipid peroxidation has been proposed to be an early event in the oxidative injury of cells22

.

An enhancement in the lipid peroxidation has been observed in Arabidopsis thalialla and cucumber coty ledons after treatment with UV -B radiation20. Takeuchi et al.23 monitored malondialdehyde formation in cucumber cotyledons and recorded higher levels of malondialdehyde after UV -B treatment. In the present investigation a direct evidence for the formation of oxyradicals after UV -B treatment has been obtained by using EPR spectroscopy.

To counteract the destructive effects of activated oxygen species, cells deploy an array of enzymatic

JAIN ef al.: UV-B STRESS AND OXYRADICALS 885

and non-enzymatic antioxidant defense. This defense system consists of low molecular weight antioxidants such as ascorbate, glutathione, a-tocopherol and ~­carotenoids24

; as well as several antioxidant enzymes such as superoxide dismutase, catalase, guaiacol peroxidase, ascorbic acid peroxidase and glutathione reductase25

•26

. Plants respond to oxidative stress by changing in vivo levels of different antioxidants which may provide protection to a particular stress14 or by inducing the activation of m-RNA transcripts of several antioxidant enzymes and the balance between generation and removal of AOS dictates the extent of damage27

•

In the present investigation the direct quenching of oxyradicals produced under UV-B stress by the antioxidants has been monitored along with the estimation of the level of antioxidants under UV-B stress.

Materials and Methods Seeds of Cucumber (Cucumis sativus L . var long

green) were obtained from Suttons and Sons Ltd. Calcutta, India. Seeds of uniform size and shape were selected, rinsed with HgCh (0.01 %), washed thoroughly under tap water and finally with distilled water. Seeds were spread on moist filter paper in Petri dishes (15 cm diam.) and allowed to grow in complete darkness at 25D±1 DC. Cotyledons were excised from seedlings grown for 48 hr in darkness.

Cotyledons from these seedlings were excised with the help of sterilized razor blade in such a way that no portion of the hypocotyl tissue remained attached to the cotyledons. The excised cotyledons were floated with their inner surface exposed in Petri dishes (9 cm diam) containing 10 ml of distilled water. All these manipulations were performed in a dark room (25 0 ± 1 DC) under a green safe lamp (Phillips 25 W covered with 8 layers of green cellophane; irradiation at the level of seedlings being 0.2 W/m2). Six cotyledons were floated per Petri dish and grown in complete darkness or exposed to UV-B. Each experiment was run with triplicate sets of cotyledons; the values presented with standard errors are the mean of three experiments.

Light source-UV-B (280-320 nrn) was obtained from FL-20-SE, Toshiba, Tokyo ("max = 311 nm; 40 W). UV-B was filtered through a polyvinyl chloride film (UV-C-O Mitsuioatsu Ltd, Japan). Irradiance at the level of seedling was 2.6 or 5.6 mW/cm2

, measured with UV-Tex a+b idm radiometer [Optix Tex. Inc., Washington D.C.].

EPR analysis-Superoxide was generated and trapped in chemical system by mixing 1 III of K02

with 100 III of phosphate buffer solution (PBS; pH 7.4, 100 ruM) and 5 III of N-t-butyl-a-phenyl-nitrone (PBN; 500 ruM). Capillaries were made from aliquot and recordings were taken.

For tissue system, six dissected (100 mg) cotyledons grown in dark or irradiated with UV-B for 1 hr were homogenized in 990 III phosphate saline buffer (PBS; pH 7.4, 100 ruM) and di-ethyl di-thio carbamic acid (DOC) 10 III (100 ruM). The homogenate was centrifuged at 10,000 rpm in a Remi R-24 centrifuge. The supernatant obtained was mixed with 5 III of PBS and 5 III of N-t-butyl-a-phenyl­nitrone (PBN; 500 ruM). The contents were vortexed gently and incubated in dark for 45 min after the addition of PBN. Dark conditions were maintained thrQughout the experiment. After the incubation, 25 III of aliquot was loaded into a quartz capillary tube and ESR spectra were recorded on an X -band EPR spectrophotometer (Varian-E-104 with TM-110 cavity).

Effect of quenchers on radical intensity-To study the effect of quenchers on the radical intensity in a chemical system, K02 (I Ill), PBS (100 rnM, 50 Ill), and PBN (500 rnM, 5 Ill) were mixed with 50 III solution of a-tocopherol, benzoquinone or ascorbic acid in desired concentration (indicated in the figures). Capillaries were made from the aliquot and spectra were recorded.

For studying the effect of quenchers on tissue system, 100 mg tissue was homogenized in PBS (PH 7.4, 100 ruM) and required concentration of 500 III tocopherol, benzoquinone or ascorbic acid was added at the time of homogenization. Contents were centrifuged and then incubated for 45 min after the addition of PBN. All the ESR spectra were recorded at room temperature under the following settings, scan range - 100 G, field set 3237 G, time constant 1 sec, scan time 16 min, modulation amplitude 2 G, modulation frequency 100 KHz, receiver gain 1.25x104xlO and 1.25x103 for tissue system and chemical system respectively, microwave power 5 mW, microwave frequency 9.01 KHz, temperature 25DC.

Antioxidant levels Ascorbic acid content-Ascorbic acid was extracted

from the control and stressed cotyledons by the method of Franke28

. Cotyledons (100 mg) were ground in mortar and pestle with a pinch of NaCl in

886 INDIAN J EXP BIOL, SEPTEMBER 2004

freshly prepared 10 m1 of meta-phosphoric acid (2%) and centrifuged at 10,000 rpm for 10 min. The supernatant was kept in dark on ice until use. Ascorbic acid was determined spectrophotometrically at 524 nm by measuring the reduction of DCPIP. 1 ml of supernatant was mixed with 1 ml distilled water, 1 ml of meta-phosphoric acid (2%), 0.5 ml of sodium citrate buffer (0.1 M, pH 2.3) and 1 m1 DCPIP (lOa mg 1-1). The reagents were added in the same order as described. Absorbance was recorded at 524 nm against blank containing water. The amount of ascorbate present was calculated with reference to a standard curve.

a-Tocopherol-It was extracted by the method of Walker and Slinger29 and estimated by the method of Pearson et al. 30

. Cotyledons (500 mg) were homogenized in 25 ml of absolute alcohol, 0.5 ml of alcoholic pyrogallol (10%) and 2-3 boiling chips. The solution was transferred toa conical flask, refluxed for 5 min, saturated aqueous-KOH (2.5 ml) was added through the condenser. The solution was again refluxed for another 5 min. The sample was cooled in an ice bath and 25 ml of cold water added along with 25 ml of petroleum ether. The solution was then transferred to a 250 ml separating funnel. The lower aqueous phase was decanted for re-extraction with 25 ml of petroleum ether. The ether fraction was collected. The solution was washed 3-4 times with

a ~

'0 ..-><

distilled water containing alcoholic pyrogallol. Petroleum ether was evaporated and the remaining matter was redissolved in a little benzene (0.2 mI). Volume was made up to 10 ml with absolute alcohol.

To 1 ml of the above-mentioned solution, 0.2% of alcoholic FeCh and 1 ml of alcoholic a,a-dipyridyl test solution (0.5%) were added. Volume was made up to 5 ml with absolute alcohol. After 10 min the absorbance was read at 520 nm. The amount of tocopherol present was calculated from the standard curve with O.l to l.0 mg tocopherol/ml.

Results Detection of oxyradicals-ESR signal of

oxyradicals produced in the cotyledons either grown in dark or exposed to UV -B (1 hr) radiations were similar to PBN-adduct formed after auto-oxidation of K02 (Fig. 1). These signals were characterized as triplet signals with hyperfine splitting of 16.2 G, which underwent a j3-hydrogen splitting 3.5 G at each of the absorption points signifying its origin from PBN-02'- adduct. UV -B exposure enhanced the intensity of the signals produced in dark grown cotyledons. The dark signal varied with the age of the cotyledons, but the enhancement with UV-B was observed at all ages (Fig. 1).

Nature of the oxyradicals-To ascertain the production of O2'- radical, superoxide dismutase

1200 o Dark IllUV-D

(5800 u :0 ~ g '0 ~400 Vl c Q)

:s

o 1--'-

24 48 72

Growth Period (hrs)

Fig. I-EPR spectra of oxyradical-PBN adduct formed at different growth periods in dark and after UV-B exposure (l hr, 5.6 mW/cm2

)

in cucumber cotyledons. a, c, e - dark controls; b, d, f - after exposure to UV-B in cotyledons isolated from 24, 48 and 72 hr. grown seedlings respectively and after auto-oxidation of KOz (g).

JAIN etal.: UV-B STRESS AND OXYRADICALS 887

a

b

-1t--~ c

Fig. 2-EPR spectra of oxyradical-PBN adduct formed from auto­oxidation of K02 [a], Fenton's ' reaction [b] and from auto­oxidation of K02 in presence of superoxide dismutase (SOD).

a

b

~ 0 ..... x

SOO

400

~ 300 ~ ~ 0 -c o 200 ~ 'iii c: 2 £:

100

d

0

[Horseradish, Sigma] was externally added to the media to convert O2'- to H20 2. A decrease in the intensity of the radical was observed by addition of SOD to K02 reaction mixture as well as to homogenates of ' cotyledons either exposed or unexposed to UV -B (Figs 2, 3). By addition of SOD a complete reduction of signal was not observed either in the chemical or in the tissue system indicating fast conversion of superoxide radical to other oxyradicals which can form the same type of adduct. The similarity of the adduct was observed by the reaction of hydroxyl radical (OH) produced by Fenton's reaction with PBN (Fig. 2). A minute difference of formation of a shoulder in the second split distinguished the OH radical adduct form that of O2'- .

Addition of SOD resulted in the reduction of Ca (66%) in the chemical system (Fig. 2); Ca (49%) in the unexposed homogenates and Ca (45%) in the cotyledons exposed to UV -B (Fig. 3).

Effect of quenchers-Many natural compounds can deactivate the oxyradicals by direct reduction, of these three compounds (benzoquinone, ascorbic acid and a-tocopherol) have been tested for their direct interaction with oxyradicals produced in the chemical system or in the cotyledons exposed to UV -B. The oxyradicals produced by auto-oxidation of K02 were quenched by all the three compounds (Figs 4, 5, 6).

Dark Treatmen

UV-B

a-SOD

ra+SOD

Fig. 3-EPR spectra of oxyradical-PBN adduct formed in dark and UV-B exposed (1 hr, 5.6 mW/cm2) cotyledons in the absence [a, c] of

superoxide dismutase (SOD) or in the presence of SOD [b, d]. Each bar represents the mean of three samples assayed in triplicates and the vertical lines indicates SE.

888 INDIAN J EXP BIOL, SEPTEMBER 2004

At the highest concentration used, the intensity of the radical was reduced by Ca (53%; benzoquinone-l mM), Ca (78%; ascorbic acid-l mM) and Ca (50%; a-tocopherol- 100 ~ (Figs 4,5,6).

~ ". 0

x,., (ij U

~t50 >. )(

0, .. 0 ~ .. c ~ oS 0>--

0 OJ, 0.' , Benzoquinone Cone (mM)

Fig. 4-EPR spectra of oxyradical-PBN adduct formed after the addition of different concentrations of benzoquinone in K02

system. I mM [a], 0.1 mM [b], 0.01 mM [c] and without the addition of benzoquinone [d]. Each bar represents the mean of three samples assayed in triplicates and the vertical lines indicates SE.

o 260

'"0 )( 200

~ :0 tlO

~ a too d 0

.~ 10

'" iii £ a D.Dl D.l

AA Concentration (mM)

Fig. 5-EPR spectra of oxyradical-PBN adduct formed after the addition of different concentrations of ascorbic acid in K02

system. I mM [a], 0.1 mM [b], om mM [c) and without the addition of ascorbic acid [d). Each bar represents the mean of three samples assayed in triplicates and the vertical lines indicates SE.

In the cotyledons, UV-B enhanced production of oxyradicals was reduced by the quenchers as indicated in the spectra (Fig. 7). Quenchers were effective on both the exposed and unexposed cotyledons and the intensity of the radicals was reduced by about 65 to 70% in the unexposed cotyledons and by about 80% in the UV-B exposed cotyledons (Fig. 8).

In vivo level of antioxidants- Endogenous levels of ascorbic acid and a-tocopherol have been estimated in the cotyledons isolated from seedlings aged for different time periods in dark and then exposed to 1 hr of UV-B. The amount of ascorbic acid varied in the cotyledons depending on the age of the seedlings from which it was isolated (Table 1). After exposure of cotyledons to UV-B radiation the endogenous level of ascorbic acid was enhanced at all the stages (Table. 1). A maximum enhancement of Ca (165%) was recorded with cotyledons isolated from seedlings grown for 24 hr in dark. In contrast to ascorbic acid the endogenous level of a-tocopherol was high in cotyledons at the initial stages of seedling growth and got reduced with the age of the seedlings (Table 1). Treatment with UV-B further reduced the level of a-tocopherol in the cotyledons (Table 2). A maximum reduction of Ca (50%) was observed in the initial growth stages.

Discussion Deleterious effects of UV -B are considered to be

due to the formation of active oxygen species20•

d

Tocopherol Cone (~M)

Fig. 6-EPR spectra of oxyradical-PBN adduct formed after the addition of different concentrations of a-tocopherol in K02

system. 100 IlM [aI, 50 J.LM [b], 25 IlM [c] and without the addition of a-tocopherol [d]. Each bar represents the mean of three samples assayed in triplicates and the vertical lines indicates SE .

JAIN et al.: UV-B STRESS AND OXYRADICALS 889

u . 500

300

200

a 100

o o 0.Q1 0.1

400 BenzOQUinone Cone. (mM)

o

o X 300

b ~

c

d

Fig. 7-EPR spectra of oxyradical-PBN adduct formed after the addition of different antioxidants in darkness [I] or after UV-B exposure [II] in cucumber cotyledons. 100 ~M a-tocopherol [a], 1 mM benzoquinone [b], 1 mM ascorbic acid [c] and without the addition of any antioxidants [d).

Oxygen can form a variety of radicals of which superoxide [02'-] and hydroxyl [OR'] radicals are the most destructive initial radicals produced. No direct evidence existed for the production of these oxyradicals in the tissues exposed to UV-B although their production under UV -B stress was suspected by various workers by monitoring the formation of MDA by lipid peroxidation8

,23. Since a direct evidence for conversion of ozone to free radicals using ESR has been established in the studies of Grimes et al.31 and Mehlhorn et al.32 and since the adverse impact of ozone and UV -B on plant growth and metabolism is either synergistic or additive, Bowler et al.8 have concluded that UV-B produces 02'-, OR' and H20 2.

The present results, using ESR spectroscopy, provide direct evidence, not obtained hitherto, for the production of O2'- and OR' radicals in the plant tissue exposed to UV-B radiations. These oxyradicals have been trapped at room temperature by using PBN and the spectra were compared with the standard chemical

'6 ~ 200 g '0 »100 ~ c: CIJ

E 0

500

~oo

300

200

100

o

o 25 50 100

Tocopherol Cone. (11AI)

o 0.01 0.1

Ascorbie acid Cone. (mM)

Fig. 8-Integrated intensity of oxyradical formed after the addition of different concentrations of benzoquinone, tocopherol and ascorbic acid to dark and UV- B (1 hr, 5.6 mW/cm2

) exposed cucumber cotyledons. Each bar represents the mean of three samples assayed in triplicates and the vertical lines indicates SE.

spectra of O2'- and OR' radicals obtained from the auto-oxidation of K02 and Fenton reaction respectively. According to Finkelstein et al. 33 a real proof of the spectrum of oxyradicals is gained by using superoxide dismutase enzyme to inhibit the signal. External application of SOD to K02 system in the present study showed dismutation of O2'- radical and reduction in the intensity of the signal. The reduced signal after addition of SOD had a visible characteristic shoulder in the second split of the three peaks indicating that this signal was due to OR' radical. A similar signal resulted when the products of Fenton reaction formed an adduct with PBN. Addition of SOD to tissue homogenates also reduced the intensity of the oxyradical-PBN adduct indicating that UV-B produced O2'- as well as hydroxyl radicals.

The production of excessive free radicals may result in oxidative damage by unwanted and excessive

890 INDIAN J EXP BIOL, SEPTEMBER 2004

Table I-Ascorbic acid and a-tocopherol content in the cotyledons of seedlings grown for different periods in darkness or after exposure to UV-B (2.6 mW/cm2)

Treatment Time­period

(hr)

o 24 48 72

[Values are mean ± SE of 3 experiments

Ascorbic acid (~g/mg FW)

a-Tocopherol (gg/mg FW)

Dark UV-B Dark UV-B

0.158±0.01 0.193±0.02 3.50±0.10 0.193±0.01 0.316±0.02 3.00±0.09 0.228±0.02 0.316±0.03 2.50±0.06 0.352±0.03 0.490±0.04 2.00±0.07

1.50±0.08 1.50±0.08 1.75±0.09 1.50±0.07

Table 2-Ascorbic acid and a-tocopherol content in the cotyledons isolated from 48 hr dark grown seedlings and exposed to different periods ofUV-B (2.6mW/cm2)

[Values are mean ± SE of 3 experiments]

Treatment Ascorbic acid a-Tocopherol UV-B (min) (~g/mgFW ) (~g/mg FW)

Control 0.193 ± 0.01 3.50 ± 0.10 15 0.230 ± 0.Q2 2.20± 0.07 30 0.316 ± 0.03 2.40 ± 0.06 45 0.253 ±0.02 2.60 ± 0.Q7 60 0.223 ± 0.02 1.50 ± 0.Q7 120 0.21O±0.01 1.10 ± 0.06

oxidation reaction. The free radicals can attack polyunsaturated fatty acids resulting in the damage of both structure and function of cell membranes in a chain reaction unless they are quenched by antioxidants34. Endogenous small molecules play an important role in the removal of toxic oxygen species because of their quenching property and they can reduce potentially destructive effect of active oxygen species 14. Amongst the several naturally occurring antioxidants present in plant tissue are ascorbic acid, a-tocopherol, glutathione and phenolic compounds.

a-Tocopherol is a principal biochemical antioxidant defense molecule against lipid peroxidation with it capacity to scavenge O2'-, OH and 102 (Ref. 35). Besides being an active in vitro chain breaking antioxidant the long chain phytol tail on a-tocopherol allow the compound to partition into lipophilic membranes of cells and organelles where it exerts its antioxidant activity in the prevention of oxidative damage36. a-Tocopherol has several possible mode of action;-(1) as a chain breaking antioxidant by trapping fatty acyl peroxyl radical; (2) as a reductant of O2'- to produce H20 2 and tocopherol quinone37; and (3) as a reactant with singlet oxygen 38. In the present study, a-tocopherol has been demonstrated to quench the oxynidicals produced in

the tissue by UV-B exposure and act as an antioxidant against UV -B induced physiological stress.

The tocopheroxyl radicals formed during the conversion of oxyradicals to hydroperoxides are reduced back to tocopherol by ascorbic acid, glutathione cycle. Thus ascorbic acid and a­tocopherol can act synergistically in the reduction of free radicals formed during any of the stress conditions39. Ascorbic acid is a water soluble antioxidant and it can rapidly react in vivo with O2'­and OH to produce water37. The present results substantiated this observation. The ability of ascorbic acid, a relatively polar acid and lipid insoluble molecule to interact directly with components of lipid bilayer has been questioned. Several investigations have suggested that oxyradicals bearing fatty acid chain may partition into hydrophilic surface of the bilayer40. Extensive hydration of the membrane surface may minimize any entropic barrier to the close juxtaposition of ascorbic acid and oxyradicals. This view is supported by the data of Scarpa et al.41

indicating direct interaction between ascorbic acid and oxyradicals in liposomes membranes.

In the results presented, benzoquinone showed to have similar effects like other antioxidants. Benzoquinone is a naturally occurring propanoid of higher plants; p-benzoquinone has been experimentally used as singlet oxygen quenching substance42. A wide variety of free and conjugated phenols exist in plants some of which have been tested as antioxidants in vivo. Ferulic acid43, thymol, carvacol and 6-gingerol have been found to decrease lipid peroxidation and effectiyely scavenge peroxy\ radicals44

• Scavenging effect of different catechins on O2'- radical generated by irradiated riboflavin system as well as free radicals generated by AAPH and DPPH has been demonstrated by using ESR spectroscopy45.

Since the antioxidants like a-tocopherol and ascorbic acid are effective quenchers of the oxyradicals, plants often respond to oxidative stress by enhancing the biosynthesis of antioxidants. An increase in the endogenous level of ascorbic acid after UV-B exposure has earlier been reported in Arabidopsis thaliana46 and wheat6. In the present results, a similar response was found in cucumber cotyledons to alleviate the detrimental effects of UV-B induced oxyradicals. The mechanism of enhancement in the level of ascorbic acid is yet to be worked out. In contrast to this, a-tocopherol level did not enhance in the cucumber cotyledons by UV-B.

JAIN et al.: UV-B STRESS AND OXYRADICALS 891

Instead, a-tocopherol content was decreased by UV­B exposure indicating its utilization in quenching oxyradicals.

In summary, the present investigation provided a direct evidence for the production of superoxide and hydroxyl radicals. Deleterious effects of these radicals was regulated by the natural quenchers like a­tocopherol, ascorbic acid and benzoquinone. The production of oxyradicals evoked a defensive system in cucumber cotyledons by way of enhanced synthesis of ascorbic acid.

Acknowledgement The work received financial support from CSIR

research grant 38(968)/99/EMR-II.

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